Mitomycin Signaling to and from p53 During the Cell Cycle; In response to DNA damage the p53 protein is post-translationally modified and promotes either cell cycle growth arrest (at both the G1/S and G2/M transitions) or apoptosis. Many wild-type p53 target genes have been identified; a few of these include GADD45, p21/Waf1, bax, 14-3-3, mdm2 and IGF-BP3. To enable students and the research community to understand more about the signaling pathways to and from p53 we will investigate the following areas. Aim 1) Provide a three month research module that enables students to grasp the complexities of p53 related cell cycle research. Aim 2) Determine if DNA damage caused by different chemotherapeutic drugs is able to induce differential activation of p53 target genes. We will examine the effects of mitomycin C (MC) and two MC derivatives. MC causes double strand breaks and the two derivatives do not. We will also compare the effects of camptothecin and Zeocin, which causes different types of DNA damage. Analysis of the gene expression level of multiple targets will be monitored using real-time RT-PCR probed with novel fluorescent probes. Aim 3) Determine if DNA damage induced differential activation of p53 target genes is cell cycle regulated. We intend to use real-time RT-PCR to examine the mRNA expression from p53 responsive genes at different times during the cell cycle in response to camptothecin, Zeocin and the mitomycins. Cell cycle fractions will be isolated using centrifugal elutriation. Aim 4) Determine if differential expression of select target gen corresponds to a difference in the in vivo and in vitro DNA binding activity of p53. Protection of the p53 binding site regions of select genes will be monitored in nuclei during differential gene activation using in vivo LM-PCR footprinting. The in vitro DNA binding activity of p53 will be examined by EMSA using nuclear extract from cultured cells and cell cycle fractions treated with DNA damaging agents. Aim 5) Examine if specific post-translational modifications of p53 are responsible for differential p53 mediated gene activation and DNA binding activity. The post-translational modification of p53 in specific drug treated fractions shown to exhibit overt differences in mRNA expression and p53 DNA binding activity will be examined by Western blot analysis using antibodies to specific phosphorylated and acetylated forms of p53. The acetylated sites that are modified will be mutated for use in the inducible p53 system.